High fat diet intake, particularly in diabetic patients, can lead to problems related to constipation, a leading cause for outpatient gastroenterological visits. Our laboratory focuses on the changes in gastrointestinal motility in diseases such as diabetes and recently on the effects of a diet high in fat. Enteric neurodegeneration can lead to intestinal dysmotility seen in slow-transit constipation, Hirschsprung's disease and diabetes. We have established a high-fat diet (HFD-34.5% kcal from fat) induced enteric neurodegeneration mouse model in our laboratory. Our preliminary data shows that mice fed a HFD have delayed gastrointestinal motility, increase TLR4 expression in enteric ganglia and excess circulating and stool lipopolysaccharide (LPS) compared to mice on a regular diet (RD). Furthermore, saturated fatty acids such as palmitate have been implicated in central neuronal toxicity. We hypothesize that altered gut microbiota in HFD fed mice in conjunction with the high palmitate levels can result in increased TLR4 signaling and consequently, drives the colonic dysmotility. To investigate the underlying mechanism(s) and the role of microbiota in HFD-induced delayed gastrointestinal motility we will feed conventional (normal and diabetic) and germ free mice a HFD for 12 weeks and assess changes in gastrointestinal motility and the enteric nervous system. We will assess the changes induced by diet of specific neuromodulatory bacterial metabolites such as GABA, histidine and glutamine. We will perform fecal transplantation experiments to determine if the HFD induced colonic motility changes can be transferred through microbiota. Finally, by modifying the intestinal bacteria with prebiotics we will attempt t ameliorate HFD induced endotoxemia and delayed gastrointestinal motility. Our data indicate that palmitate increases TLR4 expression and induces enteric neuronal apoptosis. Using primary enteric neurons and an enteric neuronal cell line, we will examine the role of TLR4-mediated Myd88 dependent signal transduction pathways in palmitate-induced neuronal apoptosis. Preliminary data indicate that HFD induces TLR4 expression and its downstream targets JNK1 (MAPK8) and C/EBP. Using enteric neural crest specific TLR4 and Myd88 knock out models we will assess the necessity and sufficiency of TLR4 in mediating HFD-induced changes in motility. The interaction of HFD on TLR4 expression and the signal transduction involved will be dissected focusing on the role of JNK1 and C/EBP? pathway. We found that HFD induces an increase in the proapoptotic miRNA, miR375 in a TLR4 dependent fashion in enteric ganglia compared to RD fed mice. We will determine the role of TLR4 in regulation of miR375 by assessing the effects of HFD- in vivo and palmitate on enteric neurons in vitro in conjunction with TLR4-/- mice. To understand the effects of HFD-induced microbial changes on miR375 we will assess miR375 expression in germ free mice fed HFD and in mice fed with or without prebiotics. Understanding the mechanisms underlying HFD-induced dysmotility and neuronal loss can help us identify new targets to treat delayed gastrointestinal motility.